The present invention relates to a method for the measurement of free thyroxine or free 3,5,3'-triiodothyronine in a liquid sample in which the thyroxine or 3,5,3'-triiodothyronine is present in both free and combined states. The method utilizes a labeled thyroxine or 3,5,3'-triiodothyronine-horseradish peroxidase conjugate which does not significantly interact with the thyroxine-binding globulin and thyroxine-binding prealbumin originally present in the sample.
It frequently is necessary to determine the concentration of free thyroxine in a liquid sample in which the thyroxine is present in both free and combined or bound states. Typically, such a determination is part of a routine hematological examination of blood serum or plasma for the prophylaxis or treatment of a variety of disorders and diseases.
Thyroxine and 3,5,3'-triiodothyronine (hereinafter, triiodothyronine) are characteristic hormones, secreted by the thyroid, which have profound physiological effects on the basal metabolic rate of mammals. While triiodothyronine exhibits greater hormonal activity than thyroxine, it appears that thyroxine is the principal circulating thyroidal hormone. Thus, the comments which follow concentrate on thyroxine but also are generally applicable to triiodothyronine.
Thyroxine is stored in the thyroid gland as thyroglobulin, a glycoprotein, and is released via proteolysis. In the blood, thyroxine is largely bound to plasma proteins, primarily thyroxin-binding globulin and thyroxine-binding prealbumin. A small but finite amount of thyroxine is free or unbound, and it is this free thyroxine which is the physiologically-active entity. Thus, the determination of free thyroxine in the blood is an important part of the diagnosis of thyroid dysfunction.
The primary problem associated with any assay for free thyroxine is the fact that free thyroxine is present in human serum at very low concentrations. The accepted normal range is from about 0.8 to about 2.5 ng. of free thyroxine per dl. of serum (8-25 pg./ml.). This range typically is much less than about 1% of the total thyroxine normally present in serum and requires accurate measurements in parts per trillion.
Free thyroxine has been determined via equilibrium dialysis, a time-consuming and operator-intensive method. In addition, there are two known immunoassays for the measurement of free thyroxine. The first (Corning Medical and Scientific Division, Corning Glass Works, Medfield, Mass.) is a two-tube method which measures the rate of transfer of thyroxine from the binding proteins to the thyroxine-specific antibody. This method, which is discussed in greater detail below, requires carefully timing the antigen pick-up by the antibody in two separate tubes. The second method (Clinical Assays Division, Travenol Laboratories, Inc., Deerfield, Ill.) employs a single tube in which the thyroxine-specific antibody is incubated with the sample for a precisely-controlled period of time; the amount of antigen picked up by the antibody is related to the free thyroxine originally present. The antibody-antigen complex is separated from the sample and then is incubated with radiolabeled thyroxine which complexes with unoccupied binding sites on the antibody. Thus, the amount of radio-activity picked up by the complex is inversely proportional to the concentration of free thyroxine originally present in the sample.
The first method above is essentially taught by U.S. Pat. No. 4,046,870 which discloses a method for determining the concentration of free thyroid hormone in a blood serum sample which comprises the steps of analyzing the sample by immunoassay (typically radioimmunoassay) for the desired thyroid hormone in the presence and absence of a blocking agent such as merthiolate (thimerosal) or 8-anilino-1-naphthalenesulfonic acid to establish a thyroid hormone binding differential and then correlating that differential with a standard curve relating the respective free thyroid hormone concentrations to binding differentials.
There still is a need, however, for a rapid, sensitive, and accurate procedure for the direct measurement of free thyroxine in a liquid sample in which the thyroxine is present in both free and combined states. Such a need now has been met through the discovery that a labeled thyroxine-horseradish peroxidase conjugate which does not significantly interact with the thyroxine-binding globulin and thyroxine-binding prealbumin originally present in the sample can be utilized in an immunoassay to permit the measurement of free thyroxine in the sample. Because the horseradish peroxidase in the conjugate substantially retains its activity when complexed with an immobilized antibody which is specific for thyroxine, the conjugate is particularly well suited for a thyroxine solid-phase enzyme immunoassay.
In general, hapten-enzyme conjugates are well known in the prior art. See, for example, U.S. Pat. Nos. 3,654,090; 3,791,932; 3,839,153; 3,850,752; 3,879,262; 4,016,043; 4,040,907; and Re. 26,169. Several of these patents are of particular interest.
U.S. Pat. No. 3,839,153 discloses an enzyme immunoassay which utilizes a double antibody approach. Thus, a hapten (or antigen)-enzyme conjugate and soluble antibody specific for the hapten (or antigen) are mixed with the sample to be tested. An insolubilized antibody then is added to the reaction mixture, wherein the insolubilized antibody is specific for the soluble antibody previously employed. The resulting insolubilized antibody-antibody-hapten (or antigen)-enzyme complex then is separated from the mixture and the enzymatic activity of either separated phase is determined. Peroxidase is included in the examples of suitable enzymes, although there is no suggestion that the procedure is suitable for the determination of thyroxine in a sample, and certainly not for free thyroxine.
U.S. Pat. No. 3,850,752 also relates to enzyme immunoassays. Briefly, the patent teaches a process for the determination of a hapten which involves adding to the sample to be tested a hapten-enzyme conjugate and an insolubilized antibody specific for the hapten. The resulting insolubilized antibody-hapten-enzyme complex then is separated from the mixture and the enzyme activity of either resulting phase is determined. Peroxidase is included in the examples of suitable enzymes and thyroxine is included in the examples of haptens which can be determined by the disclosed method. There is, however, no specific teaching of a thyroxine-peroxidase conjugate or of the determination of free hapten, e.g. thyroxine.
U.S. Pat. No. 3,879,262 also relates to enzyme immunoassays. The disclosed process actually amounts to an improvement of each of the processes of U.S. Pat. Nos. 3,839,153 and 3,850,752. Such improvement requires that the nature of the coupling between hapten and enzyme differs from the coupling between hapten and an immunogenic material, the latter conjugate being used to produce hapten-specific antibodies in an animal. Thyroxine is included in the list of suitable haptens and peroxidases are included in the list of suitable enzymes. There is no specific teaching of a thyroxine-peroxidase conjugate however. Furthermore, the disclosed process is not used to determine free hapten.
Finally, U.S. Pat. No. 4,040,907 relates to polyiodothyronine or thyroxine conjugates with enzymes. The enzymes in such conjugates must undergo substantial change in activity to be useful in enzyme immunoassays.
Two additional references are known which disclose thyroxine-peroxidase conjugates. The first is R. F. Schall, Jr. et al., Clin. Chem., 24, 1801 (1978); an abstract of the paper appeared in Clin. Chem., 24, 1033 (1978). This reference discloses a manual enzyme immunoassay for thyroxine. The procedure determines total thyroxine in serum and is carried out as follows: The serum sample is mixed with the thyroxine-horseradish peroxidase conjugate solution which also contains a blocking agent, 8-anilino-1-naphthalene-sulfonic acid, and immobilized antibody specific for thyroxine. The resulting mixture is incubated and centrifuged. The solid phase is washed and a substrate-chromogen solution is added to the solid phase. Color is allowed to develop and then is fixed. Absorbance is measured and total thyroxine content of the sample is determined from a standard curve.
The second additional reference is G. Kleinhammer et al., "Enzyme Immuno Assay for Determination of the Thyroid Binding Index", a paper presented at the annual meeting of the American Association for Clinical Chemistry, July 1978; an abstract was published in Clin. Chem., 24, 1033 (1978). According to the paper, the first attempts to develop an enzyme immunoassay able to recognize abnormal thyroxine-binding globulin (TBG) concentrations were unsuccessful, primarily because the thyroxine-horseradish peroxidase conjugate failed to bind to TBG. The assay as reported is carried out as follows: To a serum sample is added a fixed amount of thyroxine, which amount is in excess of unbound TBG in the sample. The amount of excess thyroxine not bound by TBG is measured by enzyme immunoassay using thyroxine-horseradish peroxidase and tubes coated with antibody specific for thyroxine. Thus, the procedure was not employed to determine free thyroxine in the serum. Because the trilodo-thyronine-horseradish peroxidase conjugate also failed to bind to TBG, such conjugate can be used in place of the thyroxine-horseradish peroxidase conjugate, although the latter was preferred because of the higher binding affinity of TBG for thyroxine.
It should be noted that specific enzyme immunoassays for total thyroxine are known. Such assays are based on either malate dehydrogenase or alkaline phosphatase; see, e.g., P. R. Finley and R. J. Williams, Clin. Chem., 24, 165 (1978); A. P. Jaklitsch et al., Clin. Chem., 21, 1011 (1975); F. VanLente and D. J. Fink, Clin. Chem., 24, 387 (1978); R. S. Galen and D. Forman, Clin. Chem., 23, 119 (1977); and Japanese Pat. No. 77/108017.